Enzyme Replacement Therapy (ERT) on Heart Function Changes the Outcome in Patients with Infantile-Onset Pompe Disease: A Familial History

Background Lysosomal acid alpha-glucosidase (GAA) deficiency, also known as Pompe disease, is an autosomal recessive disorder that leads to the accumulation of glycogen in lysosomes and cytoplasm, resulting in tissue destruction. Infantile-onset GAA deficiency is characterized by cardiomyopathy and severe generalized hypotonia. Without treatment, most patients die within the first two years of life. The demonstration of reduced GAA activity, followed by sequencing of the GAA gene, confirms the disease. GAA deficiency is currently treated with enzyme replacement therapy (ERT) with improved clinical outcomes and survival. Case Presentation. We describe the case of DGAA in two siblings, in which the diagnostic time point, treatment, and outcomes were completely different. The girl was diagnosed with DGAA at the age of 6 months during investigations for poor weight gain and excessive sleepiness. The finding of severe cardiomyopathy through EKG and echocardiography led to the suspicion of storage disease, and the GAA deficiency was later confirmed by genetic analysis. The girl died of complications due to the clinical picture before starting ERT. Conversely, her younger brother had the opportunity to receive an early diagnosis and the rapid onset of ERT. He is showing a regression of cardiac hypertrophy. Conclusion The advent of ERT improved clinical outcomes and survival in infantile-onset PD. Its impact on cardiac function is still under study, but different reports in the literature have shown encouraging data. Early recognition of DGAA and prompt initiation of ERT is therefore crucial to prevent the progression of the disease and improve the outcomes.


Introduction
Lysosomal acid alpha-glucosidase (GAA, also called acid maltase) defciency (Pompe disease, formerly classifed as glycogen storage disease type II (GSD II)) is a rare autosomal recessive disorder with considerable allelic heterogeneity. It is caused by pathogenic variants in the gene for GAA. Defciency of GAA (DGAA) leads to the accumulation of glycogen in lysosomes and cytoplasm, which results in tissue destruction, most afecting the skeletal muscle [1,2].
Infantile-onset DGAA is characterized by cardiomyopathy and severe generalized hypotonia. Without treatment, most patients die within the frst two years of life [3,4]. Late-onset disease (juvenile and adult presentation) is characterized by skeletal myopathy (usually in a limb-girdle distribution) and a protracted course leading to respiratory failure without cardiomyopathy [5]. Infantile-onset DGAA should be suspected in infants with profound hypotonia and cardiac insufciency [6,7]. Juvenile or adult-onset DAA should be considered in patients with progressive weakness in a limb-girdle distribution [5]. Supportive fndings may include an electrocardiogram (EKG) demonstrating a short PR interval and giant QRS complexes, suggesting biventricular hypertrophy, although this is a nonspecifc fnding (infantile-onset form) [6,4]; electromyogram demonstrating myopathic discharge, sometimes associated with abundant myotonic and complex repetitive discharges, most prominent in the paraspinal muscles (late-onset form) [5]; elevated serum creatine kinase (CK; all forms) [5][6][7]. Demonstration of reduced GAA activity in dried blood spots or leukocytes, followed by sequencing of the GAA gene, confrms the disease [6]. DGAA defciency is currently treated with enzyme replacement therapy (ERT), physical and occupational therapy, and supportive care (e.g., mechanical ventilation for respiratory failure) [8]. Te advent of ERT improved clinical outcomes and survival for both early-and late-onset DGAA [9]. Here, we describe the case of two siblings whose fate was completely diferent and infuenced by the possibility of making an early diagnosis and starting the ERT. We also ofer a brief review of the literature on the cardiac involvement of PD and on the efects of ERT.

Case 1.
A six-month-old female infant was referred to our attention for poor weight gain, poor food intake, and excessive sleepiness. Born at 40 weeks of gestational age, after a normal pregnancy, and characterized by maternal hypertension, his birth measurements were 2980 grams of weight, length 51 cm, head circumference 34 cm, and an Apgar score of 9-10. For detection of a perioral cyanotic mask occurring during and after the meal, echocardiography performed at 2 days of life showed normal parameters, apart from the size of the ventricles and septum at the upper limits of the norm. For this reason, a followup was scheduled at one month of life, which was not carried out due to noncompliance of the parents. At admission, her vital parameters were normal (SpO2 100%, HR 135 bpm, RR 47 bpm) and the clinical examination was notable for a slightly dystrophic appearance, small nose with anteverted nostrils, scaphocephaly, barrel chest, systolic murmur, axial hypotonus, and of the shoulder and pelvic girdles. Te fnding of severe left ventricular hypertrophy on EKG and a signifcant increase in CPK, GPT, and troponin I (CPK 557 U/L, CPK-MB 41.6 ng/ml, GPT 146 U/L, troponin I 0.31 ng/ml) raised the suspicion of congenital hypertrophic heart disease. A chest X-ray showed cardiomegaly and echocardiography confrmed the cardiomyopathy with the severe phenotypic expression of nonstructural hypertrophic type, with moderate reduction of the global contractile function of the left ventricle and acute heart failure ( Figure 1(a)). With suspicion of storage disease, we performed genetic tests that confrmed the diagnosis of DGAA. Unfortunately, the child died shortly after from complications related to cardiac insufciency.

Case 2.
Five years later, the parents gave birth to a boy, born at 39 weeks from spontaneous birth, with a 10-10 Apgar score and normal anthropometric measures (3.450 gr of weight, 50 cm length, and 35 cm head circumference) without any dysmorphism. Te neonatal course was characterized by hypoglycemia, hyponatremia, and hypocalcemia corrected with oral supplementation. In consideration of the family history of PD, a genetic analysis was performed which resulted positive for deletion of c.235-247 in homozygosity of exon 3 of the GAA gene. Te infant was therefore diagnosed with PD and carried out the following investigations: blood tests (Aspartate Transaminase and CK increased, 53 IU/L and 1069 IU/L, respectively), EKG, echocardiography (myocardial hypertrophy of the left ventricle, with normal kinetics and contractility, Figure 1(b)), and alpha-glucosidase assay (0.25 µmol/L/h, normal value > 1.45 µmol/L/h). To initiate the enzyme replacement therapy, the evaluation of the CRIM (crossreactive immunological material) status was also performed. Given the negative results, immunomodulatory therapy with Intravenous immunoglobulins (IVIGs) 500 mg/kg, Rituximab (12.5 mg/kg, 3 infusions), and methotrexate (0.4 mg/ kg, 5 infusions) was initiated concurrently with the frst infusions of alglucosidase alfa (Myozime, SANOFI) at starting dose of 20 mg/kg/week). Te dosage of alglucosidase alfa was then increased to 40 mg/kg/week after 2 weeks and methotrexate was replaced with sirolimus (1 mg/m 2 /die) after three months. Periodic infusions of IVIG and blood tests were warranted, along with neuro-psycho-behavioral assessments, the latter showing a substantially adequate development for age. Te child performed cadenced echocardiography, which showed a progressive improvement of the hypertrophic picture from the beginning of the ERT (Figure 1(c)). Currently, the child is 18 months old and is substantially in good health. He also passed without complications and a SARS-Cov2 infection occurred in October 2020 with febrile symptoms, but without clinically signifcant respiratory distress or cardiac sequelae.
In infantile-onset DGAA, cardiac involvement is early and often severe and is one of the most important determinants of poor prognosis. It is characterized by hypertrophic cardiomyopathy. Tus, cardiac enlargement is found in thoracic radiography. EKG fndings include short PR interval and high voltage QRS complexes in all leads because of left ventricular hypertrophy [10]. Furthermore, bradycardia in the newborn may disclose glycogenosis storage [11]. Echocardiography provides useful morphological information for the diagnosis of hypertrophic cardiomyopathy. Right and left ventricular walls are thickened [12]. Te left ventricular cavity can be reduced because of severe wall hypertrophy leading to left ventricular outfow tract obstruction and abnormal ventricular compliance [13]. Te diastolic function may be impaired because of left ventricular hypertrophy. In a Dutch patient group [14], the diastolic thickness of the left ventricular posterior wall and cardiac weight at autopsy were increased signifcantly with age. Te diagnosis of infantile-onset DGAA should be suspected in an infant with severe hypotonia and cardiac insufciency. GAA enzyme activity can be measured in white blood cells or dried blood spots. Gene sequencing is the preferred test to confrm the diagnosis since it is routinely available, is less invasive, may provide genotype-phenotype information, and may help predict cross-reactive immunologic material (CRIM) status (amount of residual endogenous GAA production) in some cases [6].
Te diferential diagnosis for classic infantile disease with hypertrophic cardiomyopathy includes the following theories: (i) Lysosome-associated membrane protein 2 defciency, presenting with hypertrophic cardiomyopathy, muscle weakness, and hypotonia (ii) Fatty acid oxidation disorders, including very longchainacyl-CoA dehydrogenase defciency, longchain 3-hydroxy-acyl-CoA dehydrogenase defciency, carnitine transporter defciency, carnitineacylcarnitine translocase defciency, and carnitine palmitoyltransferase defciency type 2, presenting in infancy with hypertrophic cardiomyopathy with nonketotic hypoglycemia (iii) Mitochondrial and respiratory chain disorders, which may present with hypotonia, cardiomyopathy, hepatomegaly, and seizures (iv) Other infantile-onset hypotonia without cardiomyopathy, including spinal muscular atrophy type 1 and GSD type IIIa Without treatment, most patients with the classic infantile form have unremitting deterioration with death during the frst two years of age from cardiac insufciency [7]. However, prolonged survival has been reported in infants with less severe cardiomyopathy [7].
Te current treatment for DGAA is ERT with alglucosidase alfa. Standard dosing is 20 mg/kg given intravenously every two weeks. Dosing may be increased twofold to 20 mg/kg once a week or 40 mg/kg every two weeks in those with a poor response to initial therapy. A multidisciplinary care team is needed to obtain short-and long-term improvements in cardiac and skeletal muscle function and survival [9,[15][16][17].
New treatments such as gene therapy are under development to increase the intrinsic ability of the afected cells to produce GAA. Key components of gene therapy strategies include the choice of vector promoter and the route of administration. Te efcacy of gene therapy depends on the ability of the vector to drive gene expression in the target tissue and on the recipient's immune tolerance to the transgene protein [18].
As noted above, cardiac involvement is one of the most important determinants of the poor prognosis of infantileonset DGAA. However, diferent literature data show that ERT is slowly changing the prognosis. A long-term followup study of 17 patients with infantile-onset DGAA (up to 5.4 to 12 years of age) on ERT demonstrated improvements in cardiac measures of left ventricular mass index within fve months of initiation of ERT [15]. Tis study also reported improvements in gross motor function, although residual muscle weakness with contractures, dysphagia with aspiration risk, hypernasal speech, and osteopenia were still present. In a study, 13 infants born in Taipei from 2010 through 2015 were diagnosed with infantile DGAA by a nationwide newborn screening program [16]. Te mean age at the start of ERT was 12 days. Left ventricular function improved after three to four months of therapy. Institution of ERT has been reported as early as 18 hours after birth with resolved hypertrophic cardiomyopathy and normal neurodevelopment at 46 weeks [19]. Taken together, these data, along with our case, clearly show that cardiac hypertrophy associated with DGAA disease is a reversible phenomenon following the early onset of ERT. Further studies and the continuation of the followup of these patients are necessary to establish whether these results are stable over time and to evaluate further problems these children will encounter.

Conclusions
DGAA is an inherited storage disorder burdened by a very poor prognosis in untreated infantile-onset children. It is characterized by cardiomyopathy and severe generalized hypotonia, and the median age at death is nearly 8 months without treatment. Te advent of ERT has improved clinical outcomes and survival. Its impact on cardiac function is still under study, but diferent reports in the literature are showing encouraging data. Hypertrophy of the myocardium, already found in the neonatal period, seems to be reversible with the onset of ERT, although it is currently impossible to establish whether these efects are long-lasting or not. However, early recognition of these conditions and prompt initiation of ERT is crucial to prevent progression of the disease and improve the outcomes.

Data Availability
All data generated or analyzed during this study are included in this article and its supplementary information fles. Te data supporting this manuscript are from previously reported studies and datasets, which have been cited. Te processed data are available in the supplementary fle and from the corresponding author upon request.

Ethical Approval
Tis study adhered to the ethical guidelines for medical and health research involving human subjects established by the government of Italy.

Consent
Written consent for the publication of data and images was obtained from the parents of the patients.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
ML, KR, IM, and LI contributed to the conception and design. ML, KR, MEG, and IM contributed to the acquisition and interpretation of data. LI revised and edited the manuscript. All authors drafted the article and approved its fnal version.